CIENCIA 11(2), 113-118, 2003 Maracaibo, Venezuela The essential oil of Coleus amboinicus Loureiro chemical composition and evaluation of insect anti-feedant effects Dilexa Valera1, Roimar Rivas1, Jorge Luis Avila2, Lianne Aubert2, Miguel Alonso-Amelot2 and Alfredo Usubillaga1* 1Facultad de Farmacia, Universidad de Los Andes, Mérida, Venezuela. 2 Grupo de Química Ecológica, Facultad de Ciencias, Universidad de Los Andes, Mérida, Venezuela

Recibido: 12-09-02 Aceptado: 30-05-03

Abstract The chemical composition of the essential oil of Coleus amboinicus Loureiro, an aromatic plant with ethnobotanical uses, has been analyzed by gas chromatography coupled with mass spectrometry and its insect antifeedant properties examined using Sitophilus oryzae and Tribo- lium castaneum, two important stored grain insect pests. Two samples were collected, one at Rancherías (1.100 meters above sea level) and at the Medicinal Plants Garden, Faculty of Phar- macy, Mérida (1400 m.a.s.l.). Carvacrol and p-cymene were found to compose most of the volatile material, 74.5% (Mérida) and 74.1% (Rancherías). The oil from Mérida, was found to contain a larger proportion of carvacrol (64.7%) and sesquiterpenes (17.4%) than the oil from Rancherias (55.3% and 10.5%). On the other hand the oil from Rancherías was richer in p-cymene (18.8% against 9.8%) and monoterpenes. The essential oil in large dosage (1.25%) on hardened flour feeding disks was found unable to induce any negative response in the feeding behavior of the test insects, nor was any mortality recorded within the 60 h of exposure of the bioassays. Key word: Coleus amboinicus Loureiro; essential oil; carvacrol; p-cymene, Tribolium Castaneum; Sitophilus oryzae. El aceite esencial del Coleus amboinicus Loureiro composición química y evaluación de sus efectos anti-alimentarios en insectos

Resumen La composición química del aceite esencial de Coleus amboinicus Loureiro, una planta aromática con usos etnobotánicos, ha sido analizada mediante cromatografía de gases acopla- da con espectrometría de masas, así como sus propiedades antialimentarias contra los coleóp- teros Sitophilus oryzae y Tribolium castaneum, dos insectos plaga de importancia en los cerea- les. Se colectaron dos muestras, una en Rancherías (1100 m.s.n.m.) y otra en el Jardín de Plan- tas Medicinales de la Facultad de Farmacia en Mérida (1400 m.s.n.m.). Se encontró que el Car- vacrol y el p-cymene constituían la porción mayoritaria del aceite esencial, 74,5% (Mérida) y 74,1% (Rancherías). El aceite proveniente de Mérida posee una mayor proporción de carvacrol

* Autor para la correspondencia. E-mail: [email protected]

Scientific Journal from the Experimental Faculty of Sciences, at La Universidad del Zulia, Volume 11 Nº 2, April-June 2003 114 The essential oil of Coleus amboinicus Loureiro

(64,7%) y sesquiterpenos (17,4%) que el aceite de Rancherías (55,3% y 10,5%). Por otra parte el aceite proveniente de Rancherías es más rico en p-cimeno (18,8% vs 9,8%) y monoterpenos. Este aceite esencial en altas dosis (1,25%) sobre discos endurecidos de harina no fue capaz de inducir respuesta negativa alguna en el comportamiento alimentario de los insectos de prueba, así como tampoco hubo mortalidad durante las 60 horas de exposición que duraron los bioen- sayos. Palabras clave: Aceite esencial; carvacrol; Coleus amboinicus Loureiro; p-cymene; Sitophilus oryzae; Tribolium castaneum.

Introduction C. amboinicus and to test its anti-feedant properties on Tribolium castaneum and Sito- Coleus amboinicus Lour (Lamiaceae), philus oryzae two important stored grain in- popularly known as “Orégano orejón”, is an sect pests. aromatic shrub widely distributed in Vene- zuela. It is used to treat colds and cough as Materials and Methods well as arthritic inflammations (1). Accord- ing to the Missouri Botanical Gardens the Plant Material name Plectranthus amboinicus is a synonym of C. amboinicus. Its insect repellent proper- Leaves of C. amboinicus were collected ties have been tested (2) and at least another at Rancherías, Campo Elías Municipality, member of the Coleus genus, C. aromaticus, Mérida State, at 1.200 meters above sea has been found to cause reduction in egg level and at the Medicinal Plants Garden laying capacity, retard in adult emergence (1400 m. asl), Faculty of Pharmacy, Univer- and weight loss in the pulse beetle Calloso- sity of Los Andes, Merida. Voucher speci- bruchus maculatus F (3). A moderate allelo- mens (A.Usubillaga 24a and 24b) have been pahic effect of the powdered leaves of C. am- deposited at the MERF Herbarium, Faculty boinicus against the water hyacinth is also of Pharmacy; University of Los Andes. on record (4). Studies performed in India demonstrated the “fungistatic” properties of Insects the essential oil of this plant (5), but studies Malathion-resistant strain of red flour on its essential oil composition have not beetles Tribolium castaneum (Herbst) from a been reported. Brieskorn and Riedel (6) iso- 13-year old collection at Grupo de Química lated eight triterpenic acids from the leaves Ecológica, was cultivated in ½ liter glass jars of C. amboinicus and later they reported the using a mixture of whole wheat flour and isolation of six flavones, one flavanone and wheat germ (5%) as growth medium at 28 ± one flavonolol (7). No further phytochemical 1°C. Rice weevils Sitophilus oryzae (L.) from studies of this plant have been reported but a 15 year old collection of the same labora- a review (8) describes the components and tory were grown in ½ liter glass jars in whole medicinal importance of plants belonging to wheat, barley or maize grains from local different Coleus species. The essential oil of suppliers. C. aromaticus has been studied by Baslas et al (9) and plectrin, an aphid antifeedant Extraction of the oil and measurement diterpene has been isolated from Plectran- of its physical constants thus barbatus (10). Considering the record The plant material (1.35 Kg and 1.1 Kg of some Coleus species, it was decided to respectively) was cut and hydrodistilled in a study the composition of the essential oil of Clevenger-type apparatus for 3 h yielding a

Scientific Journal from the Experimental Faculty of Sciences, at La Universidad del Zulia, Volume 11 Nº 2, April-June 2003 D. Valera et al. / Ciencia Vol. 11, Nº 2 (2003) 113-118 115 colorless oil. The oil was dried over anhy- Bioassays drous sodium sulfate and stored under ni- Thin 0.5 cm2 hardened flour disks were trogen at 4°- 6°C. Optical rotations were prepared from a mixture of wheat flour and measured in a Jasco DIP 370 electropolari- water with 2% butter added to prevent meter at 23°C; density with a picnometer cracking followed by mild heating (40°C) for and refractive index with an Abbe refracto- 1 h in a ventilated oven. Treatment disks meter at 25°C. were dosaged with 20 µL solutions of essen- tial oil of C. amboinicus in acetone with the Analysis aid of a microsyringe to yield 500 µg of sol- 2 GC analyses were performed using a ute per cm of flour disk (1.25% w/w). Con- Perkin-Elmer AutoSystem gas chroma- trol disks were dosaged with pure acetone. tograph equipped with FID detector and data Treatment and control feed were ventilated handling system. A 5% phenylmethyl polysi- under mild heating until all traces of solvent loxane fused-silica column (HP-5, 30 m, 0.25 had been removed. Disks and insects were mm i.d., film thickness 0.25 µm) was used. weighted before and after bioassays. For one Column oven temperature was programmed way assay, ten unsexed 7 -12 day old adult from 60 to 200°C at 4°C/min, subsequently insects were placed ina8cmPetri dish con- at 8°C/min up to 280°C. The injector and de- taining one treated disk and placed in the tector temperatures were 200°C and 280°C, dark for 60 h, after which the remaining food respectively. The carrier gas was helium at was removed and weighted. There were five 0.8 mL/min. The samples (1.0 µL) were in- replicates per experiment. From the weight jected using a split ratio of 1:100. Retention change in insect and disk masses the follow- indices were calculated relative to C8-C24 al- ing parameters were calculated (13): feeding kanes. The percentage composition of the preference index Fi, amount of disk and oils were calculated by the normalization compound devoured, insect mass gained or method from the GC peak areas. lost during feeding, and feed conversion effi- ciency into biomass (ECI). For comparison of GC-MS analysis was carried out on a the means the Kurkal-Wallis and Tuckey Model 5973 Hewlett Packard GC-MS sys- non parametric tests (P= 0.001) were em- tem fitted with a HP-5MS fused-silica col- ployed using Statistix V 4.0 from Analytical umn (30 m x 0.25 mm i.d., film thickness Software Inc., Minneapolis, Minesota. 0.25 µm). Oven temperature program was the same used for the HP-5 column for GC analysis; the transfer line temperature Results and Discussion was held at 280°C; source temperature, Table 1 presents density, optical rota- 230°C; quadrupole temperature, 150°C; tion, refractive indices, and essential oil carrier gas, helium, adjusted to a linear ve- yields obtained from leaves of C. amboinicus locity of 34 m/s; ionization energy, 70 eV; collected at Rancherías and at the Faculty of scan range, 10–500 amu; 2.2 scans/s. Pharmacy, Mérida. Samples (1.0 µL) were injected as 2% solu- tions of the oils in n-heptane. A Hewlett The results of the analyses of the vola- Packard ALS injector was used with split tile components from the leaves of C. ambo- ratio 1:100. The identity of the oil compo- inicus are listed in Table 2 in order of elution nents was established from their GC reten- on the HP-5 column. The identified compo- tion indices (11, 12), by comparison of nents represented 99.6% of the oil from their MS spectra with those of standard Rancherías and 99.8% of the oil from the compounds available in the laboratory, Faculty of Pharmacy Garden. The most and by library search (Wiley 6 th ed.). abundant constituent in both cases was carvacrol (55.3% and 64.7%), followed by p-

Scientific Journal from the Experimental Faculty of Sciences, at La Universidad del Zulia, Volume 11 Nº 2, April-June 2003 116 The essential oil of Coleus amboinicus Loureiro

Table 1 Yields and physical properties of essential oil obtained from leaves of Coleus amboinicus collected at Rancherías and at the Faculty of Pharmacy, University of Los Andes, Merida

Site of Collection Oil yield Density Optical Refractive %v/w g/mL Rotation Index Rancherías 0.22 0.9241 -2.4° 1.5075 Faculty of Pharmacy 0.20 0.8894 -1.8° 1.4903 cymene (18.8% and 9.8%) and g-terpinene (7.2% and 4.7%). The sesquiterpene fraction Table 2 made up 10.5% of the oil from Rancherías, Percentage composition of the essential oil from where t-caryophyllene (5.4%) and a-berg- the leaves of Coleus amboinicus Loureiro amotene (3.3%) were the most abundant collected at Rancherías and at the Faculty of compounds while the oil from the Faculty of Pharmacy, Merida Pharmacy contained a larger proportion of sesquiterpenes (17.4%) but t-caryophyllene KI Compound % oil from % oil (9.1%) and a-bergamotene (4.6%) were also Rancherias from the most abundant constituents of that Mérida fraction. It is interesting to point out that the 920 a-thujene 0.4 – sum of the aromatic monoterpenes carvac- 927 a-pinene 0.2 – rol and p-cymene was practically the same 949 camphene 1.0 – (74.1% and 74.5%) for both oils. Rancherías 981 1-octen-3-ol 1.0 0.8 is located at an altitude of 1100 m while the 993 b-myrcene 1.1 0.3 Faculty of Pharmacy of the University of Los a Andes is at 1400 m, and both places are not 1001 -felandrene 0.1 – d more than 10 Km apart but the weather is 1009 3-carene 0.1 – quite different in both locations. Rancherias 1015 a-terpinene 1.3 0.6 is a rather dry place while at the Garden of 1022 p-cymene 18.8 9.8 the Faculty of Pharmacy the weather is hu- 1038 b-felandrene 0.6 – mid most of the time. This difference in 1053 g-terpinene 7.2 4.7 weather conditions would explain the differ- 1064 c-sabinene 0.2 – ences in oil composition. hydrate Sitophilus oryzae and Tribolium casta- 1080 terpinolene 0.2 – neum provided two feeding strategies among 1095 t-sabinene 0.1 – the stored cereal insect pests. The former is hydrate a primary seed predator as it bores the ker- 1161 borneol 0.1 – nell shell with powerful mandibles located 1172 terpin-4-ol 0.9 1.0 at the tip of the elongated feeding apparatus. 1300 thymol 0.5 0.5 Only when the pericarp and mesocarp have 1310 carvacrol 55.3 64.7 been perforated the insect begins feeding. By contrast, T. castaneum is a secondary 1421 t-caryophyllene 5.4 9.1 grain pest that profits from bores practised 1433 a-bergamotene 3.3 4.6 by primary seed predators, flour and pow- 1453 a-humulene 1.3 2.6 dered meal derived therefrom. Therefore, 1582 caryophyllene 0.4 1.1 the two insects provide a wide spectrum of oxide feeding strategies used by the many cereal 1625 di-elapiol 0.1 – grain insect species. In addition, their expo-

Scientific Journal from the Experimental Faculty of Sciences, at La Universidad del Zulia, Volume 11 Nº 2, April-June 2003 D. Valera et al. / Ciencia Vol. 11, Nº 2 (2003) 113-118 117 sure to hardened flour disks whose surface the body weight did not show any added in- have been treated with purportedly obnox- crement from the excess feed ingested. This ious chemicals from Coleus amboinicus and is reflected in the conservative values of the other plant derived essential oils allows to ECI or efficiency of conversion of ingested test the repellency as well as feeding inhibi- food. Hence, no adverse physiological reac- tion properties of the material. tion with influence on body weight gain could be detected. It may be concluded, Tables 3 and 4 collect the effects of C. therefore that carvacrol and p-cymene, the amboinicus oil from two sources in Mérida major components of C. amboinicus oil, are State on the feeding behavior of the two in- innocuous to these insects at the large dos- sect species. In the large doses employed age presented. The repellency effects re- (1.25% v/w) there was no difference in the corded earlier (2) do not apply to the stored feeding response of S. oryzaerelative to con - grain insects tested here. trols and no noticeable after-effect upon the ingestion of as much as 10 µg of the oil per Conclusions insect. On its part, T. castaneum showed a slight increase in the phagoactivity of the Carvacrol and p-cymene were found to treated disk relative to the control although compose about 74% of the essential oil of Coleus amboinicus. This oil did not induce

Table 3 One way assay feeding parameters of Sitophilus oryzae exposed to feed treated with 1.25% (w/w) of C. amboinicus essential oil from plants collected in two localities of Mérida State; (a): Medicinal Plants, garden of the Faculty of Pharmacy, Universidad de Los Andes (1400 m asl) and (b) Campo Elías Municipality, 1200 m asl. Equal letters denote statistical undifferentiation (N = 5, 10 individuals per replicate)

Sample Consumption/ins Consumption Ingested ess. Oil Mortality (%) ect (mg) ±SD relative to control (µg/insect) ±SD (%) Oil (a) 0.744 ± 0.043 (c) 96.9 10615 ± 1172 (c) 0 Oil (b) 0.669 ± 0.132 (c) 87.0 8001 ± 1809 (c) 0 Control 0.768 ± 0.032 (c) 100.0 0 0

Table 4 One way assay feeding parameters of Tribolium castaneum obtained under the same conditions as described in the legend of Table 2. DW represents the insect body weight change relative to the unfed individual during the same time period, determined in a parallel assay. Equal letters denote statistical undifferentiation (N = 5, 10 individuals per replicate)

Sample Consumption/in Consumption Ingested ess. Oil DW/insect (mg) % weight ECI±SD sect (mg) ± SD relative to (µg/insect) ± SD ±SD change control (%) Oil (a) 0.428 ± 0.046c 118.2 6339 ± 1158e 0.04 ± 0.009f 2 0.321 ± 0.027 g Oil (b) 0.452 ± 0.059c,d 124.9 6435 ± 0.788e 0.04 ± 0.01 f 2 0.315 ± 0.044g Control 0.362 ± 0.085c 100 0 0.04 ± 0.01 f 2 0.397 ± 0.067g

Scientific Journal from the Experimental Faculty of Sciences, at La Universidad del Zulia, Volume 11 Nº 2, April-June 2003 118 The essential oil of Coleus amboinicus Loureiro any negative response in the feeding behav- 6. BRIESKORN C.H., RIEDELW. Arch Pharm ior of Tribolium castaneum or Sitophilus 310: 910-916, 1977. oryzae. 7. BRIESKORN C.H., RIEDEL W. Planta Me- dica 31: 308-310, 1977. Acknowledgements 8. BASLAS R.K., KUMAR P. Herba Hung 20: The authors are grateful to FONACIT, 213-221, 1981. grant S1-97001302 for providing partial 9. BASLAS R.K., KUMAR P. J Indian Chem funding of this research. Soc 58: 103-104, 1981. 10. KUBO I., MATSUMOTO T., TORI M., References ASAKAWA Y. Chem Lett 1513-1516, 1984. 1. GIL-OTAIZA R. Plantas usuales en la me- 11. JENNINGS W., SHIBAMOTO T. Qualita- dicina popular venezolana Edición tive Analysis of Flavor and Fragrance C.D.C.H.T-ULA (Mérida), pp. 52-54, 1997. Volatiles by Glass Capillary Gas Chro- 2. GOMEZ-RODRIGUEZ A. Margarita: Me- matography, Academic Press New York (USA), pp. 469, 1980. dicina popular, Ediciones de la Federa- ción Farmacéutica Venezolana, Serie 3 (Ca- 12. ADAMS R.P. Identification of Essential racas), Vol. 1, pp.119, 1982. Oil Components by Gas Chromatogra- 3. KATHIRESAN R.M. Crop Protection 19 phy/Mass Spectrometry, Allured Publish- (8-10): 705-708, 2000. ing Corp. Carol Stream, IL (USA), pp. 469, 1995. 4. BABU A., RAJA N., ALBERT S., IGNACI- MUTHU S., DORN S. Biol Agric Hort 17 13. ALONSO-AMELOT M., AVILA J.L., OTERO (2): 145-150, 1999. LD., MORA F., WOLF B. Journal of Chemi- cal Ecology 20: 1161-1177, 1994. 5. SAMUEL C.O., SRIVASTAVA L., TRIPATHI S. Indian Journal Plant Prot 23: 174- 179, 1995.

Scientific Journal from the Experimental Faculty of Sciences, at La Universidad del Zulia, Volume 11 Nº 2, April-June 2003